Cells in a population are in many aspects unique in their characteristics, even in a seemingly homogenous culture or tissue. Gene expression levels show large cell to cell variations, due to external (extrinsic) and internal (intrinsic) sources of factors Likewise, when exposed to identical stimuli, cells often behave stochastically. This means that data obtained from a population of cells cannot be assumed to reflect the behavior of the individual cell. It has been suggested that cells can respond to stimuli by bursts in transcriptional activity and operate as a binary switch; that is in an all-or-none fashion.
Usually, gene expression on the RNA level is monitored on a routine basis by a multi-step procedure. First, the respective cellular sample is removed from the culture vessel. In case of adherent cells harvesting may be supported by trypsination (treatment with a Trypsin-EDTA solution) in order to detach the adherent cell from the solid support. Secondly, the collected cells are pelleted and subjected to cell lysis. As a third step it is usually required to at least partially purify the RNA or mRNA that is present in the sample (EP 0 389 063). Afterwards, a first strand cDNA synthesis step is performed with an RNA dependent DNA polymerase such as AMV (Roche Applied Science Cat. No: 11 495 062) or MMuLV (Roche 11 062 603) Reverse Transcriptase.
Subsequently, the amount of generated cDNA is quantified either by means of quantitative PCR (Sagner, G., and Goldstein, C., BIOCHEMICA 3 (2001) 15-17) or, alternatively by means of amplification and subsequent hybridization onto a DNA microarray (Kawasaki, E. S., Ann. N.Y. Acad. Sci. 1020 (2004) 92-100). In case of PCR, a one step RT-PCR may be performed, characterized in that the first strand cDNA synthesis and subsequent amplification are catalyzed by the same Polymerase such as T.th Polymerase (Roche Applied Science Cat. No. 11 480 014).
In traditional real time RT-PCR or qRT-PCR, RNA is first isolated from cells in a time consuming procedure that can lead to a loss of material. Using the CellsDirect cDNA Synthesis System (Invitrogen Cat No. 11737-030), the cells are lysed and the cDNA is generated from the lysate in a single tube with minimal handling and no sample loss. DNase I is added to eliminate genomic DNA prior to first-strand synthesis. After synthesis, the first-strand cDNA can be transferred directly to the qPCR reaction without intermediate organic extraction or ethanol precipitation. This kit has been optimized for small cell samples, ranging from 10,000 cells down to a single cell. A similar protocol is disclosed in (WO 08/135,197).
In this context, the technical problem underlying the present invention was to provide a high throughput method and a kit that allow for a further simplified gene expression monitoring analysis protocol.